Fluorescence microscopy is a mature and widely used tool for studying biological systems at the microscopic level. Due to the diffraction of light, all fluorescence microscopy techniques are limited to a lateral resolution of approximately 250 nm as given by 0.61*lambda/NA, where lambda and NA refer to the wavelength of light and numerical aperture of microscope objective. However, there exist many biological features comprised of structures significantly smaller than 250 nm requiring a resolution that exceeds the diffraction limit. Single molecule localization microscopy (SMLM) represents one such exemplary method for achieving sub-diffraction limited resolution. Two closely related variants of SMLM include STochastic Optical Reconstruction Microscopy (STORM) and Photo-Activated Localization Microscopy (PALM). All SMLM techniques function similarly whereby an image of a sparse subset of photochemically activated fluorophores is collected and subsequent localization of each fluorophore performed by fitting a suitable point spread function to each observed Any disk. Activated fluorophores are then photochemically deactivated or switched from the emissive (active) to dark (inactive) state and back again to the emissive state. This defines one switching cycle of the fluorophore which is performed repeatedly until a large enough number of fluorophores has been imaged and localized to yield the desired spatial resolution of the final image. Typical lateral fluorophore localization accuracy is on the order of 25 nm which ultimately allows for a final image spatial resolution of approximately 50 nm.
All SMLM techniques are predicated on three requirements. First, fluorophores used to image the sample must be of the group comprising those which are capable of blinking. That is, they must be capable of transitioning between emissive and dark states under typical laboratory conditions. Secondly, the population distribution of emissive to dark fluorophores must be controllable via some means. Typically this is accomplished photochemically through the use of two lasers operating a different wavelengths; one to quench fluorescence and the other to activate the emissive state. Lastly, all SMLM techniques rely on the ability to efficiently localize the emitting fluorophores to a high degree of precision and accuracy. It is the last of these three requirements that is the focus of this disclosure.
Despite the advantages SMLM brings to the researcher, most organelles and cellular structures cannot be fully resolved without high-resolution along all three dimensions. Historically, 3D microscopy is performed using a scanning confocal microscope, however, lateral and axial resolution are then again limited by diffraction effects. Extending the capabilities of SMLM techniques to the third, axial, dimension overcomes these problems. Various techniques have previously been reported that allow for 3D SMLM (Huang et al. Science Vol 319; Piestun and Quirin US 2011/0249866 A1). 3D SMLM techniques rely on optically modifying the point spread function (PSF) of the microscope in such a manner that introduces a transverse variation that changes predictably and rapidly with axial displacement. One such method is to incorporate a cylindrical lens into the optical path of the microscope which introduces transverse astigmatism aberration into the PSF. A two dimensional elliptical Gaussian function with variable transverse widths may be used to model the aberrated PSF and extract depth information.
SMLM requires thousands of images to be acquired in order to generate a sufficient number of localization points so that the final reconstructed image may have adequate spatial resolution. This restricts the temporal resolution of any SMLM technique to the time required to localize enough fluorophores such that the distance between any two adjacent localized points is less than half the distance of the desired spatial resolution. The only two ways of improving temporal resolution are by increasing the rate at which fluorophores are switched from the active to inactive state or by increasing the number density of activated fluorophores in a given image. Increasing the fluorophore switching rate requires higher activation laser energy and results in increased photodamage to the sample. Increasing the number density of activated fluorophores per image leads to significant overlap of fluorescent spots (Airy disks) which, when using previously disclosed methods (Piestun), results in decreased localization accuracy and ultimately a degradation in the final image's spatial resolution.
This disclosure is directed to improving the speed of fluorophore localization in SMLM techniques in three dimensions by allowing significantly greater number density of activated fluorophores per image while maintaining high localization precision and accuracy